Compositions comprising an inhibitor of lysine specific demethylase-1

ABSTRACT

Described herein are amorphous and crystalline forms of pharmaceutically acceptable salts of the lysine specific demethylase-1 inhibitor 4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile. Also described are pharmaceutical compositions suitable for administration to a mammal that include the lysine specific demethylase-1 inhibitor, and methods of using the lysine specific demethylase-1 inhibitor for treating diseases or conditions that are associated with lysine specific demethylase-1 activity.

RELATED APPLICATION

This Application is a continuation of U.S. application Ser. No.16/032,002, filed Jul. 10, 2018, now U.S. Pat. No. 10,316,016, which isa continuation of U.S. application Ser. No. 15/793,512, filed Oct. 25,2017, now U.S. Pat. No. 10,047,069, which is a continuation of U.S.application Ser. No. 15/344,426, filed Nov. 4, 2016, now U.S. Pat. No.9,828,358, which claims priority benefit of U.S. Provisional PatentApplication No. 62/251,507, filed Nov. 5, 2015, the contents of theseapplications are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

Described herein is the lysine specific demethylase-1 inhibitor4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,and pharmaceutically acceptable salts, solvates, and crystalline formsthereof.

BACKGROUND OF THE INVENTION

A need exists in the medicinal arts for an effective treatment of cancerand neoplastic disease. Lysine specific demethylase-1 has beenimplicated in a number of diseases or conditions, such as breast cancer,lung cancer, prostate cancer, glioblastoma, and leukemia, as well asothers diseases or conditions.

SUMMARY OF THE INVENTION

Described herein is4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,pharmaceutically acceptable salts, pharmaceutically acceptable solvates(including hydrates), polymorphs, and amorphous phases thereof, andmethods of use thereof.

In one aspect, described herein is a pharmaceutically acceptable salt of4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,wherein the pharmaceutically acceptable salt is in crystalline form. Insome embodiments, the pharmaceutically acceptable salt is besylate salt,wherein the pharmaceutically acceptable salt is in crystalline form.

In one aspect, described herein is a crystalline Form 1 of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt that is characterized as having:

-   -   (a) an X-Ray powder diffraction (XRPD) pattern substantially the        same as shown in FIG. 1;    -   (b) an X-ray powder diffraction (XRPD) pattern with        characteristic peaks at 4.9° 2-Theta, 9.7° 2-Theta, 13.4°        2-Theta, 18.0° 2-Theta, 18.5° 2-Theta;    -   (c) a DSC thermogram with an endotherm having an onset        temperature at about 317° C.;    -   (d) a DSC thermogram substantially similar to the one set forth        in FIG. 2; or    -   (e) combinations thereof.

Also described herein is a pharmaceutical composition comprising acrystalline form of a pharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,and at least one additional ingredient selected from pharmaceuticallyacceptable carriers, diluents and excipients. In some embodiments, thepharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrileis a besylate salt. In some embodiments, the pharmaceutical compositionis in a form suitable for oral administration to a mammal. In someembodiments, the pharmaceutical composition is in an oral solid dosageform. In some embodiments, the pharmaceutical composition comprisesabout 0.5 mg to about 200 mg of crystalline4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt.

In some embodiments, described herein is a pharmaceutical compositioncomprising a crystalline form of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt as described herein, and at least one additionalingredient selected from pharmaceutically acceptable carriers, diluentsand excipients. In some embodiments, the pharmaceutical compositionincludes Form 1 of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzo-nitrilebesylate salt. In some embodiments, the pharmaceutical composition is ina form suitable for oral administration to a mammal. In someembodiments, the pharmaceutical composition is in an oral dosage form.In some embodiments, the pharmaceutical composition is in an oral soliddosage form. In some embodiments, the pharmaceutical composition is inthe form of a tablet, pill, or capsule. In some embodiments, thepharmaceutical composition is in the form of a capsule. In someembodiments, the pharmaceutical composition is in the form of a tablet.In some embodiments, the pharmaceutical composition is in the form of amoisture barrier coated tablet. In some embodiments, the pharmaceuticalcomposition comprises about 0.5 mg to about 200 mg of crystalline4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt. In some embodiments, the pharmaceutical compositioncomprises about 0.5 mg to about 200 mg of crystalline4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt.

Also provided is an article of manufacture comprising multiple unitdoses of the oral solid dosage form pharmaceutical composition describedherein in a high-density polyethylene (HDPE) bottle equipped with ahigh-density polyethylene (HDPE) cap. In some embodiments, high-densitypolyethylene (HDPE) bottle further comprises an aluminum foil inductionseal and silica gel desiccant.

Also described herein is4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt that is in amorphous form. Also described herein is apharmaceutical composition comprising amorphous4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt, and at least one additional ingredient selected frompharmaceutically acceptable carriers, diluents and excipients. In someembodiments, the pharmaceutical composition is in a form suitable fororal administration to a mammal. In some embodiments, the pharmaceuticalcomposition is in an oral solid dosage form.

In one aspect, described herein is the use of a crystalline form of apharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilein the treatment of cancer in a mammal. In another aspect, describedherein is the use of crystalline4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt in the treatment of cancer in a mammal. In another aspect,described herein is the use of amorphous4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt in the treatment of cancer in a mammal. In someembodiments, the cancer is amenable to treatment with a lysine specificdemethylase-1 inhibitor. In some embodiments, the cancer is breastcancer, lung cancer, prostate cancer, glioblastoma, or leukemia.

In certain embodiments described herein, a crystalline form of apharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrileis used in the manufacture of a medicament for the treatment orprevention of diseases, disorders, or conditions associated with lysinespecific demethylase-1 activity.

Also described is a method of treating cancer in a mammal comprisingadministering to the mammal a crystalline pharmaceutically acceptablesalt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile.In some embodiments, the crystalline pharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrileis the besylate salt. In some embodiments, the cancer is breast cancer,lung cancer, prostate cancer, glioblastoma, or leukemia.

Also provided is the use of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt for the manufacture of a medicament for the treatment orprevention of cancer in a human. Further provided is the use of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt for the manufacture of a medicament for the treatment orprevention of cancer in a human wherein the cancer is breast cancer,lung cancer, prostate cancer, glioblastoma, or leukemia. In someembodiments,4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt is crystalline.

Also described herein are processes for the preparation of crystallineform of a pharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile.

Also described herein are processes for the preparation of crystalline4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt. The disclosed processes provide for the preparation ofcrystalline4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt in good yield and high purity.

Other objects, features and advantages of the methods and compositionsdescribed herein will become apparent from the following detaileddescription. It should be understood, however, that the detaileddescription and the specific examples, while indicating specificembodiments, are given by way of illustration only, since variouschanges and modifications within the spirit and scope of the inventionwill become apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the XRPD of Form 1 of crystalline4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt.

FIG. 2 illustrates the DSC thermogram of Form 1 of crystalline4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt.

FIG. 3 illustrates the XRPD of amorphous crystalline4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt.

DETAILED DESCRIPTION

Epigenetics is the study of heritable changes in gene expression causedby mechanisms other than the underlying DNA sequence. Molecularmechanisms that play a role in epigenetic regulation include DNAmethylation and chromatin/histone modifications.

The genomes of eukaryotic organisms are highly organized within thenucleus of the cell. Tremendous compaction is required to package the 3billion nucleotides of the human genome into the nucleus of a cell.Chromatin is the complex of DNA and protein that makes up chromosomes.Histones are the major protein component of chromatin, acting as spoolsaround which DNA winds. Changes in chromatin structure are affected bycovalent modifications of histone proteins and by non-histone bindingproteins. Several classes of enzymes are known which modify histones atvarious sites.

There are a total of six classes of histones (HI, H2A, H2B, H3, H4, andH5) organized into two groups: core histones (H2A, H2B, H3, and H4) andlinker histones (HI and H5). The basic unit of chromatin is thenucleosome, which consists of about 147 base pairs of DNA wrapped aroundthe core histone octamer, consisting of two copies each of the corehistones H2A, H2B, H3, and H4.

Basic nucleosome units are then further organized and condensed by theaggregation and folding of nucleosomes to form a highly condensedchromatin structure. A range of different states of condensation arepossible, and the tightness of chromatin structure varies during thecell cycle, being most compact during the process of cell division.

Chromatin structure plays a critical role in regulating genetranscription, which cannot occur efficiently from highly condensedchromatin. The chromatin structure is controlled by a series of posttranslational modifications to histone proteins, notably histones H3 andH4, and most commonly within the histone tails which extend beyond thecore nucleosome structure. These modifications are acetylation,methylation, phosphorylation, ribosylation, sumoylation, ubiquitination,citrullination, deimination, and biotinylation. The core of histones H2Aand H3 can also be modified. Histone modifications are integral todiverse biological processes such as gene regulation, DNA repair, andchromosome condensation.

Histone methylation is one of the most important chromatin marks, theseplay important roles in transcriptional regulation, DNA-damage response,heterochromatin formation and maintenance, and X-chromosomeinactivation. A recent discovery also revealed that histone methylationaffects the splicing outcome of pre-mRNA by influencing the recruitmentof splicing regulators. Histone methylation includes mono-, di-, andtri-methylation of lysines, and mono-, symmetric di-, and asymmetricdi-methylation of arginines. These modifications can be either anactivating or repressing mark, depending on the site and degree ofmethylation.

Histone Demethylases

A “demethylase” or “protein demethylase,” as referred to herein, refersto an enzyme that removes at least one methyl group from polypeptide.Demethylases comprise a JmjC domain, and can be a methyl-lysine ormethyl-arginine demethylase. Some demethylases act on histones, e.g.,act as a histone H3 or H4 demethylase. For example, an H3 demethylasemay demethylate one or more of H3K4, H3K9, H3K27, H3K36 and/or H3K79.Alternately, an H4 demethylase may demethylate histone H4K20.Demethylases are known which can demethylate either a mono-, di- and/ora tri-methylated substrate. Further, histone demethylases can act on amethylated core histone substrate, a mononucleosome substrate, adinucleosome substrate and/or an oligonucleosome substrate, peptidesubstrate and/or chromatin (e.g., in a cell-based assay).

The first lysine demethylase discovered was lysine specific demethylase1 (LSD1/KDM1), which demethylates both mono- and di-methylated H3K4 orH3K9, using flavin as a cofactor. A second class of Jumonji C (JmjC)domain containing histone demethylases were predicted, and confirmedwhen a H3K36 demethylase was found used a formaldehyde release assay,which was named JmjC domain containing histone demethylase 1(JHDM1/KDM2A).

More JmjC domain-containing proteins were subsequently identified andthey can be phylogenetically clustered into seven subfamilies: JHDM1,JHDM2, JHDM3, JMJD2, JARID, PHF2/PHF8, UTX/UTY, and JmjC domain only.

LSD-1

Lysine-specific demethylase 1 (LSD1) is a histone lysine demethylasethat specifically demethylates monomethylated and dimethylated histoneH3 at K4 and also demethylates dimethylated histone H3 at K9. Althoughthe main target of LSD1 appears to be mono- and di-methylated histonelysines, specifically H3K4 and H3K9, there is evidence in the literaturethat LSD1 can demethylate methylated lysines on non-histone proteinslike p53, E2F1, Dnmt1 and STAT3.

LSD1 has a fair degree of structural similarity and amino acididentity/homology to polyamine oxidases and monoamine oxidases, all ofwhich (i. e., MAO-A, MAO-B and LSD1) are flavin dependent amine oxidaseswhich catalyze the oxidation of nitrogen-hydrogen bonds and/ornitrogen-carbon bonds. LSD1 also includes an N-terminal SWRIM domain.There are two transcript variants of LSD1 produced by alternativesplicing.

In some embodiments,4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrileis capable of inhibiting LSD1 activity in a biological sample bycontacting the biological sample with4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile.In some embodiments,4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrileis capable of modulating the level of histone 3 lysine 4 methylation inthe biological sample. In some embodiments,4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrileis capable of modulating histone-3 lysine-9 methylation levels in thebiological sample.

One embodiment provides a method of treating cancer in a patient in needthereof, comprising administering to the patient the compound4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,or a pharmaceutically acceptable salt thereof. One embodiment provides amethod of treating cancer in a patient in need thereof, comprisingadministering to the patient the compound4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,or a pharmaceutically acceptable salt thereof. One embodiment provides amethod of treating cancer in a patient in need thereof, comprisingadministering to the patient a composition comprising4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,or a pharmaceutically acceptable salt thereof. Another embodimentprovides a method of treating cancer in a patient in need thereof,comprising administering to the patient a composition comprising4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate. In a further embodiment is the method for treating cancer in asubject wherein the cancer is selected from breast cancer, lung cancer,prostate cancer, glioblastoma, and leukemia. In a further embodiment isthe method for treating cancer in a subject wherein the cancer isselected from acute myeloid leukemia (AML), acute lymphoblastic leukemia(ALL), small cell lung cancer (SCLC), non-small cell lung cancer(NSCLC), neuroblastoma, small round blue cell tumors, glioblastoma, orER⁻ breast cancer.

4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrile

The term“4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluoro-benzonitrile”refers to the compound with the following structure:

4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrileis described in U.S. patent application Ser. No. 14/701,304.

Pharmaceutically acceptable salts of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrileinclude, but are not limited to, acid addition salts, formed by reactingthe compound with a pharmaceutically acceptable inorganic acid, such as,for example, hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, metaphosphoric acid, and the like; or with an organicacid, such as, for example, acetic acid, propionic acid, hexanoic acid,cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid,malonic acid, succinic acid, malic acid, maleic acid, fumaric acid,trifluoroacetic acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonicacid, toluenesulfonic acid, 2-naphthalenesulfonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, butyric acid,phenylacetic acid, phenylbutyric acid, valproic acid, and the like.

In some embodiments, described herein is a pharmaceutically acceptablesalt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile.In some embodiments, described herein is a pharmaceutically acceptablesalt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrile,wherein the pharmaceutically acceptable salt is a p-toluene sulfonicacid salt (also known as tosylate), sulfuric acid salt, methanesulfonicacid salt (also known as mesylate), benzenesulfonic acid salt (alsoknown as besylate), phosphoric acid salt, or benzoic acid salt. In someembodiments, described herein is a pharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,wherein the pharmaceutically acceptable salt is the p-toluene sulfonicacid salt. In some embodiments, described herein is a pharmaceuticallyacceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrile,wherein the pharmaceutically acceptable salt is the sulfuric acid salt.In some embodiments, described herein is a pharmaceutically acceptablesalt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrile,wherein the pharmaceutically acceptable salt is the methanesulfonic acidsalt. In some embodiments, described herein is a pharmaceuticallyacceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrile,wherein the pharmaceutically acceptable salt is the benzenesulfonic acidsalt. In some embodiments, described herein is a pharmaceuticallyacceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,wherein the pharmaceutically acceptable salt is the phosphoric acidsalt. In some embodiments, described herein is a pharmaceuticallyacceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrile,wherein the pharmaceutically acceptable salt is the benzoic acid salt.

Amorphous Phase

In some embodiments,4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt is amorphous. In some embodiments, the amorphous phase of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt has an XRPD pattern showing a lack of crystallinity.

Form 1

In some embodiments,4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt is crystalline. In some embodiments, described herein is acrystalline Form 1 of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt. In some embodiments, Form 1 of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt is characterized as having:

-   -   (a) an X-Ray powder diffraction (XRPD) pattern substantially the        same as shown in FIG. 1;    -   (b) an X-ray powder diffraction (XRPD) pattern with        characteristic peaks at 4.9° 2-Theta, 9.7° 2-Theta, 13.4°        2-Theta, 18.0° 2-Theta, 18.5° 2-Theta;    -   (c) a DSC thermogram with an endotherm having an onset        temperature at about 317° C.;    -   (d) a DSC thermogram substantially similar to the one set forth        in FIG. 2; or    -   (e) combinations thereof.

In some embodiments, Form 1 has an X-Ray powder diffraction (XRPD)pattern substantially the same as shown in FIG. 1.

In some embodiments. Form 1 has an X-ray powder diffraction (XRPD)pattern with characteristic peaks at 4.9° 2-Theta, 9.7° 2-Theta, 13.4°2-Theta, 18.0° 2-Theta, 18.5° 2-Theta.

In some embodiments, Form 1 has substantially the same X-ray powderdiffraction (XRPD) pattern post storage at 40° C. and 75% RH for atleast a week.

In some embodiments, Form 1 has substantially the same X-ray powderdiffraction (XRPD) pattern post storage at 25° C. and 96% RH for atleast a week.

In some embodiments, Form 1 has a DSC thermogram with an endothermhaving an onset temperature at about 317° C.

In some embodiments, Form 1 has a DSC thermogram substantially similarto the one set forth in FIG. 2.

In some embodiments, Form 1 is characterized as having properties (a),(b), (c), and (d), or any combination thereof

Preparation of Crystalline Forms

In some embodiments, a crystalline form of a pharmaceutically acceptablesalt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate, is prepared as outlined in the Examples. It is noted thatsolvents, temperatures and other reaction conditions presented hereinmay vary.

Suitable Solvents

Therapeutic agents that are administrable to mammals, such as humans,must be prepared by following regulatory guidelines. Such governmentregulated guidelines are referred to as Good Manufacturing Practice(GMP). GMP guidelines outline acceptable contamination levels of activetherapeutic agents, such as, for example, the amount of residual solventin the final product. Preferred solvents are those that are suitable foruse in GMP facilities and consistent with industrial safety concerns.Categories of solvents are defined in, for example, the InternationalConference on Harmonization of Technical Requirements for Registrationof Pharmaceuticals for Human Use (ICH), “Impurities: Guidelines forResidual Solvents, Q3C(R3) (November 2005).

Solvents are categorized into three classes. Class 1 solvents are toxicand are to be avoided. Class 2 solvents are solvents to be limited inuse during the manufacture of the therapeutic agent. Class 3 solventsare solvents with low toxic potential and of lower risk to human health.Data for Class 3 solvents indicate that they are less toxic in acute orshort-term studies and negative in genotoxicity studies.

Class 1 solvents, which are to be avoided, include: benzene; carbontetrachloride; 1,2-dichloroethane; 1,1-dichloroethene; and1,1,1-trichloroethane.

Examples of Class 2 solvents are: acetonitrile, chlorobenzene,chloroform, cyclohexane, 1,2-dichloroethene, dichloromethane,1,2-dimethoxyethane, N,N-dimethylacetamide, N,N di-methylformamide,1,4-dioxane, 2-ethoxyethanol, ethyleneglycol, formamide, hexane,methanol, 2-methoxyethanol, methylbutylketone, methylcyclohexane,N-methylpyrrolidine, nitromethane, pyridine, sulfolane, tetralin,toluene, 1,1,2-trichloroethene and xylene.

Class 3 solvents, which possess low toxicity, include: acetic acid,acetone, anisole, 1-butanol, 2-butanol, butyl acetate, tert-butylmethylether (MTBE), cumene, dimethyl sulfoxide, ethanol, ethyl acetate, ethylether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropylacetate, methyl acetate, 3-methyl-1-butanol, methylethylketone,methylisobutylketone, 2-methyl-1-propanol, pentane, 1-pentanol,1-propanol, 2-propanol, propyl acetate, and tetrahydrofuran.

In some embodiments, compositions comprising a crystalline form of apharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate, include a residual amount of an organic solvent(s). In someembodiments, compositions comprising a crystalline form of apharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrilebesylate, include a detectable amount of an organic solvent(s). In someembodiments, compositions comprising a crystalline form of apharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate, include a residual amount of a Class 3 solvent. In someembodiments, the organic solvent is a Class 3 solvent. In someembodiments, the Class 3 solvent is selected from the group consistingof acetic acid, acetone, anisole, 1-butanol, 2-butanol, butyl acetate,tert-butylmethyl ether, cumene, dimethyl sulfoxide, ethanol, ethylacetate, ethyl ether, ethyl formate, formic acid, heptane, isobutylacetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol,methylethyl ketone, methylisobutyl ketone, 2-methyl-1-propanol, pentane,1-pentanol, 1-propanol, 2-propanol, propyl acetate, and tetrahydrofuran.In some embodiments, the Class 3 solvent is ethanol.

The methods and compositions described herein include the use of acrystalline form of a pharmaceutically acceptable salt4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate. In addition, the crystalline forms of the pharmaceuticallyacceptable salt of4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate, can exist in unsolvated as well as solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike.

Certain Terminology

The term “cancer” as used herein refers to an abnormal growth of cellswhich tend to proliferate in an uncontrolled way and, in some cases, tometastasize (spread).

The terms “treat,” “treating” or “treatment,” as used herein, includealleviating, abating or ameliorating at least one symptom of a diseaseor condition, preventing additional symptoms, inhibiting the disease orcondition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, delaying progression of condition, relieving a conditioncaused by the disease or condition, or stopping the symptoms of thedisease or condition either prophylactically and/or therapeutically. Insome embodiments, treatment includes extending progression-freesurvival. In some embodiments, treatment includes reducing the relativerisk of disease progression compared to other treatment options.

The term “progression-free survival” is the amount of time during andafter the treatment of a disease, such as cancer, that a patient liveswith the disease but it does not get worse. In a clinical trial,measuring progression-free survival is one technique to determine theefficacy of the treatment.

The term “pharmaceutically acceptable excipient,” as used herein, refersto a material, such as a carrier, diluent, stabilizer, dispersing agent,suspending agent, thickening agent, etc. which allows processing theactive pharmaceutical ingredient (API) into a form suitable foradministration to a mammal. In one aspect, the mammal is a human.Pharmaceutically acceptable excipients refer to materials which do notsubstantially abrogate the desired biological activity or desiredproperties of the compound (i.e., API), and is relatively nontoxic,i.e., the material is administered to an individual without causingundesirable biological effects or interacting in a deleterious mannerwith any of the components of the composition in which it is contained.

“Active pharmaceutical ingredient” or API refers to a compound thatpossesses a desired biological activity or desired properties. In someembodiments, an API is a crystalline form of a pharmaceuticallyacceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate. In some embodiments, the API is crystalline4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate. In some embodiments, the API has a purity of greater than 90%,greater than 95%, greater than 96%, greater than 97%, greater than 98%,greater than 98%, or greater than 99%.

The term “pharmaceutical composition” refers to a mixture of acrystalline form of a pharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate, with other chemical components, such as carriers, stabilizers,diluents, dispersing agents, suspending agents, thickening agents,excipients, etc. The pharmaceutical composition facilitatesadministration of the compound to a mammal.

“Detectable amount” refers to an amount that is measurable usingstandard analytic methods (e.g., ion chromatography, mass spectrometry,NMR, HPLC, gas chromatography, elemental analysis, IR spectroscopy,inductively coupled plasma atomic emission spectrometry, USP<231>MethodII, etc.) (ICH guidances, Q2A Text on Validation of AnalyticalProcedures (March 1995) and Q2B Validation of Analytical Procedures:Methodology (November 1996)).

The term “acceptable” with respect to a formulation, composition oringredient, as used herein, means having no persistent detrimentaleffect on the general health of the subject being treated.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an agent being administeredwhich will relieve to some extent one or more of the symptoms of thedisease or condition being treated. The result can be reduction and/oralleviation of the signs, symptoms, or causes of a disease, or any otherdesired alteration of a biological system. For example, an “effectiveamount” for therapeutic uses is the amount of the composition comprisinga compound as disclosed herein required to provide a clinicallysignificant decrease in disease symptoms. The term “therapeuticallyeffective amount” includes, for example, a prophylactically effectiveamount. The effective amount will be selected based on the particularpatient and the disease level. It is understood that “an effect amount”or “a therapeutically effective amount” varies from subject to subject,due to variation in metabolism of drug, age, weight, general conditionof the subject, the condition being treated, the severity of thecondition being treated, and the judgment of the prescribing physician.In one embodiment, an appropriate “effective” amount in any individualcase is determined using techniques, such as a dose escalation study.

The terms “kit” and “article of manufacture” are used as synonyms.

The term “modulate,” as used herein, means to interact with a targeteither directly or indirectly so as to alter the activity of the target,including, by way of example only, to enhance the activity of thetarget, to inhibit the activity of the target, to limit the activity ofthe target, or to extend the activity of the target. The term “subject”or “patient” encompasses mammals. In one aspect, the mammal is a human.

Pharmaceutical Compositions/Formulations

Pharmaceutical compositions are formulated using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which are used pharmaceutically. Suitable techniques,carriers, and excipients include, but are not limited to, those foundwithin, for example, Remington: The Science and Practice of Pharmacy,Nineteenth Ed (Easton, Pa.: Mack Publishing Co., 1995); Hoover, John E.,Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.1975); Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms (Marcel Decker, New York, N.Y., 1980); and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999), herein incorporated by reference in their entirety.

In some embodiments, a crystalline form of a pharmaceutically acceptablesalt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate, is formulated for oral administration to a mammal. In someembodiments, a crystalline form of a pharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate, is formulated into an oral dosage form. In some embodiments, acrystalline form of a pharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate, is formulated into a solid oral dosage form. In someembodiments, a crystalline form of a pharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate, is formulated into a tablet, powder, pill, capsule, and thelike, for oral ingestion by a mammal.

Contemplated pharmaceutical compositions provide a therapeuticallyeffective amount of a crystalline form of a pharmaceutically acceptablesalt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile besylate, enabling, forexample, once-a-day, twice-a-day, three times a day, etc.administration. In one aspect, pharmaceutical compositions provide aneffective amount of a crystalline form of a pharmaceutically acceptablesalt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrilebesylate, enabling once-a-day dosing.

Dose Amounts

In one embodiment, the daily dosages appropriate for a crystalline formof a pharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate, is from about 1 to about 30 mg/kg per body weight.

Kits/Articles of Manufacture

For use in the therapeutic methods of use described herein,kits/articles of manufacture are also described herein. Such kitsinclude a carrier, package, or container that is optionallycompartmentalized to receive one or more doses of a pharmaceuticalcomposition of a crystalline form of a pharmaceutically acceptable saltof4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate, for use in a method described herein. The kits provided hereincontain packaging materials. Packaging materials for use in packagingpharmaceutical products include, but are not limited to those describedin e.g., U.S. Pat. No. 5,323,907. Examples of pharmaceutical packagingmaterials include, but are not limited to, blister packs, bottles,tubes, bags, containers, bottles, and any packaging material suitablefor a selected formulation and intended mode of administration andtreatment. A wide array of formulations of a crystalline form of apharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluoro-benzonitrilebesylate, and compositions thereof are contemplated, as are a variety oftreatments for any disease, disorder, or condition that would benefit bytreatment with an LSD-1 inhibitor.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In one embodiment, a label is on or associated with the container. Inone embodiment, a label is on a container when letters, numbers or othercharacters forming the label are attached, molded or etched into thecontainer itself; a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a compound provided herein. The pack, for example, containsmetal or plastic foil, such as a blister pack. In one embodiment, thepack or dispenser device is accompanied by instructions foradministration. In one embodiment, the pack or dispenser is alsoaccompanied with a notice associated with the container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the drug for human or veterinary administration.Such notice, for example, is the labeling approved by the U.S. Food andDrug Administration for prescription drugs, or the approved productinsert. In one embodiment, compositions containing a crystalline form ofa pharmaceutically acceptable salt of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrile,such as4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate, formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

EXAMPLES

Methods

X-Ray Powder Diffraction (XRPD)

Bruker AXS C2 GADDS

1 X-Ray Powder Diffraction patterns were collected on a Bruker AXS C2GADDS diffractometer using Cu Kα radiation (40 kV, 40 mA), automated XYZstage, laser video microscope for auto-sample positioning and a HiStar2-dimensional area detector. X-ray optics consists of a single Göbelmultilayer mirror coupled with a pinhole collimator of 0.3 mm.

The beam divergence, i.e., the effective size of the X-ray beam on thesample, was approximately 4 mm. A θ-θ continuous scan mode was employedwith a sample-detector distance of 20 cm that gives an effective 2θrange of 3.2°-29.70. Typically, the sample was exposed to the X-ray beamfor 120 seconds. The software used for data collection was GADDS forXP/2000 4.1.43 and the data were analyzed and presented using DiffracPlus EVA v15.0.0.0.

Ambient Conditions

-   -   All samples (damp or dry) were run under ambient conditions as        flat specimens. Approximately 1-2 mg of the sample was lightly        pressed on a glass slide to obtain a flat surface.

Non-Ambient Conditions (Variable Temperature Experiments)

-   -   Samples run under non-ambient conditions were mounted on a        silicon wafer with heat-conducting compound. The sample was then        heated to the appropriate temperature at 10° C./min and        subsequently held isothermally for 1 minute before data        collection was initiated.        Bruker AXS D8 Advance

High resolution X-Ray Powder diffraction patterns were collected on aBruker D8 diffractometer using Cu Kα radiation (40 kV, 40 mA), 0-20goniometer, and divergence of V4 and receiving slits, a Ge monochromatorand a Lynxeye detector. The instrument is performance checked using acertified Corundum standard (NIST 1976). The software used for datacollection was Diffrac Plus XRD Commander v2.6.1 and the data wereanalyzed and presented using Diffrac Plus EVA v15.0.0.0.

Samples were run under ambient conditions as flat plate specimens usingpowder as received. The sample was gently packed into a cavity cut intopolished, zero-background (510) silicon wafer. The sample was rotated inits own plane during analysis. The details of the data collection are:

-   -   Angular range: 2 to 42° 2θ    -   Step size: 0.05° 2θ    -   Collection time: 0.5 s/step        Nuclear Magnetic Resonance (NMR)

NMR spectra were collected on a Bruker 400 MHz instrument equipped withan auto-sampler and controlled by a DRX400 console. Automatedexperiments were acquired using ICON-NMR v4.0.7 running with Topspinv1.3 using the standard Bruker loaded experiments. Samples were preparedin DMSO-d₆ and off-line analysis was carried out using ACD SpectrusProcessor 2012.

Differential Scanning Calorimetry (DSC)

DSC data were collected on a Mettler DSC 823E equipped with a 34position auto-sampler. The instrument was calibrated for energy andtemperature using certified indium. Typically, 0.9-6 mg of each sample,in a pin-holed aluminum pan, was heated at 10° C./min from 25° C. to350° C. A nitrogen purge at 50 ml/min was maintained over the sample.The instrument control and data analysis software was STARe v12.1.

Thermo-Gravimetric Analysis (TGA)

TGA data were collected on a TA Instruments Q500 TGA, equipped with asixteen-position auto-sampler. The instrument was temperature calibratedusing certified Alumel and Nickel. Typically, 8 mg-11 mg of each samplewas loaded onto a pre-tared aluminum DSC pan and heated at 10° C./minfrom ambient temperature to 350° C. A nitrogen purge at 60 ml/min wasmaintained over the sample. The instrument control software wasAdvantage for Q Series v2.5.0.256 and Thermal Advantage v5.5.3 and thedata were analyzed using Universal Analysis v4.5A.

Water Determination by Karl Fischer Titration (KF)

The water content of each sample was measured on a Metrohm 874 OvenSample Processor at 150° C. with 851 Titrano Coulometer using HydranalCoulomat AG oven reagent and nitrogen purge. Weighed solid samples wereintroduced into a sealed sample vial. Approx 10 mg of sample was usedper titration and duplicate determinations were made. Data collectionand analysis using Tiamo v2.2.

Chloride Content Determination by Titrimetric Analysis

The method for the analysis of chloride is by oxygen flask combustion ofthe sample. Once the combustion and absorption into solution hasoccurred, the samples were titrated using a calibrated Mercuric Nitratesolution. The samples are analyzed together with a blank and organicanalytical standard reagents to ensure accuracy. The accuracy with thismethod is ±0.3% absolute with a detection limit of 0.10%.

Chemical Purity Determination by HPLC

Purity analysis was performed on an Agilent HP1100 series systemequipped with a diode array detector and using ChemStation softwarevB.04.03 using the method detailed below:

TABLE 1 HPLC method for chemical purity determinations Parameter ValueSample ~0.3 mg/ml in acetonitrile:water 1:1 Preparation Column SupelcoAscentis Express C18, 100 × 4.6 mm, 2.7 μm Column 25 Temperature (° C.)Injection (μl) 3 Wavelength, 255, 90 Bandwidth (nm) Flow Rate 2 (ml/min)Phase A 0.1% TFA in water Phase B 0.085% TFA in acetonitrile TimetableTime (min) % Phase A % Phase B 0 95 5 6 5 95 6.2 95 5 8 95 5Gravimetric Vapor Sorption (GVS)

Sorption isotherms were obtained using a SMS DVS Intrinsic moisturesorption analyzer, controlled by DVS Intrinsic Control software v1.0.1.2(or v 1.0.1.3). The sample temperature was maintained at 25° C. by theinstrument controls. The humidity was controlled by mixing streams ofdry and wet nitrogen, with a total flow rate of 200 ml/min. The relativehumidity was measured by a calibrated Rotronic probe (dynamic range of1.0-100% RH), located near the sample. The weight change, (massrelaxation) of the sample as a function of % RH was constantly monitoredby the microbalance (accuracy ±0.005 mg).

Approximately 13 mg of sample was placed in a tared mesh stainless steelbasket under ambient conditions. The sample was loaded and unloaded at40% RH and 25° C. (typical room conditions). A moisture sorptionisotherm was performed as outlined below (two scans giving one completecycle). The standard isotherm was performed at 25° C. at 10% RHintervals over a 0%-90% RH range. Data analysis was carried out usingMicrosoft Excel using DVS Analysis Suite v6.2 (or 6.1 or 6.0). Thesample was recovered after completion of the isotherm and re-analyzed byXRPD.

TABLE 2 Method for SMS DVS Intrinsic experiments Parameter ValueAdsorption - Scan 1 40-90 Desorption/Adsorption - Scan 2 90-0, 0-40Intervals (% RH) 10 Number of Scans 4 Flow rate (ml/min) 200 Temperature(° C.) 25 Stability (° C./min) 0.2 Sorption Time (hours) 6 hour time outpKa Determination and Prediction

Data were collected on a Sirius GLpKa instrument with a D-PASattachment. Measurements were made at 25° C. in methanol/water mixturesby potentiometry. The titration media was ionic-strength adjusted (ISA)with 0.15 M KCl (aq). The values found in the methanol/water mixtureswere corrected to 0% co-solvent via a Yasuda-Shedlovsky extrapolation.The data were refined using Refinement Pro software v2.2.3. Predictionof pKa values was made using ACD/Labs Percepta 2012.

Log P Determination and Prediction

Data were collected by potentiometric titration on a Sirius GLpKainstrument using three ratios of octanol: ionic-strength adjusted (ISA)water to generate Log P, Log Pion, and Log D values. The data wererefined using Refinement Pro software v2.2.3. Prediction of Log P valueswas made using ACD/Labs Percepta 2012.

Thermodynamic Solubility in FeSSiF

Solubility was determined by suspending sufficient compound in fed statesimulated intestinal fluid (FeSSIF) to give a maximum finalconcentration of >3 mg/ml of the parent free-form of the compound. Thesuspension was equilibrated at 25° C. for 24 hours then the pH wasmeasured. The suspension was then filtered through a glass fibre Cfilter. The residue was analysed by XRPD as a damp solid. The filtratewas diluted by an appropriate factor prior to analysis by HPLC.Quantitation by HPLC was performed with reference to a standard solutionof approximately 0.2 mg/ml in DMSO. Different volumes of the standard,diluted and undiluted sample solutions were injected. The solubility wascalculated using the peak areas determined by integration of the peakfound at the same retention time as the principal peak in the standardinjection. Analysis was performed on an Agilent HP1100 series systemequipped with a diode array detector and using ChemStation softwarevB.04.03.

TABLE 3 HPLC method for thermodynamic solubility measurements ParameterValue Type of method Reverse phase with gradient elution ColumnPhenomenex Luna, C18 (2) 5 μm 50 × 4.6 mm Column 25 Temperature (° C.)Standard 1, 2, 3, 5, 7, 10 Injections (μl) Test Injections (μl) 1, 2, 3,10, 15, 20 Detection: 255, 90 Wavelength, Bandwidth (nm) Flow Rate(ml/min) 2 Phase A 0.1% TFA in water Phase B 0.085% TFA in acetonitrileTimetable Time (min) % Phase A % Phase B 0.0 95 5 1.0 80 20 2.3 5 95 3.35 95 3.5 95 5 4.4 95 5Ion Chromatography (IC)

Data were collected on a Metrohm 861 Advanced Compact IC using IC Netsoftware v2.3. Accurately weighed samples were prepared as stocksolutions in an appropriate dissolving solution and dilutedappropriately prior to testing. Quantification was achieved bycomparison with standard solutions of known concentration of the ionbeing analyzed.

TABLE 4 IC method for anion chromatography Parameter Value Type ofmethod Anion exchange Column Metrosep A Supp 5 - 250 (4.0 × 250 mm)Column Temperature (° C.) Ambient Injection (□l) 10 DetectionConductivity detector Flow Rate (ml/min) 0.7 Eluent 3.2 mM sodiumcarbonate; 1.0 mM sodium hydrogen carbonate in a 5% acetone aqueoussolution.

Example 1: Salt Study of4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrile

An investigation of salt forms was performed to discover crystalline,non-hygroscopic salt forms of4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrilesuitable for use as a pharmaceutical product.

Characterization of Free Base and Monohydrochloride Salt

The4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilefree base is crystalline and has HPLC purities >96%, showed a DSCendotherm at 203.5° C. (ΔH=−89 J/g) and a solubility in FeSSIF of 0.28mg/ml. DSC, TGA and KF analyses suggests this material contains looselyadsorbed water.

4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilehydrochloride was a weak crystalline monohydrochloride (HCl Form 1). Thepresence of amorphous content in the sample was inferred from thepresence of a halo in the XRPD diffractogram and the possiblerecrystallization event in the DSC thermogram. The material washygroscopic with a 14% uptake at 90% RH by GVS. A reversible hysteresisat 60%-90% RH by GVS was associated with the formation of a new hydrateform. Partial crystallization of a new form after 14 days storage at 40°C./75% RH was confirmed by XRPD. DSC endotherm at 290.6° C. (ΔH=−84 J/g)coincides with the onset of degradation, thus the endotherm is notindicative of a pure sample melt. HPLC purity of 97.6% for thehydrochloride salt was comparable to the freebase while FeSSIFsolubility was higher at 0.75 mg/ml.

Materials

Commercial chemicals and solvents were purchased from Aldrich or Fluka.Acid stock solutions used in the screen were made up as described inTable 5:

TABLE 5 Stock solutions used in salt screen 1.1 eq for 30 mg ofCounter-ion Concentration Solvent API (μl) p-Toluene sulfonic 1.0M THF73 acid—pTSA Sulfuric acid—SO4 1.0M THF 73 Methanesulfonic 1.0M THF 73acid—MSA Benzenesulfonic 1.0M THF 73 acid—BSA Phosphoric 1.0M THF 73acid—PHOA Malonic acid—MLNA 1.0M THF 73 L-Tartaric acid—TAR 1.0M THF 73Fumaric acid—FUA 0.5M Methanol:THF (1:1) 146 Citric acid—CA 1.0M THF 73L-Malic acid—MA 1.0M THF 73 Benzoic acid—BA 1.0M IPA 73 Succinicacid—SUCA 1.0M Methanol 73Salt Feasibility Assessment

Solubility assessment of the material was performed as follows:4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilefree base (10 mg) was treated with increasing volumes of solvent untilthe material fully dissolved or until a maximum of 70 vol had been used.After each addition of solvent, the system was gently heated to 50° C.and then allowed to stand at room temperature for 5 min before theaddition of a new aliquot of solvent.

To assess the feasibility for salt formation in different solvents, thesamples from the solubility assessment were treated with 1.1 eq ofp-Toluenesulfonic acid (25 μL of 1M acid solution in THF) at 50° C.,held for 30 min then cooled down to 5° C. at 0.1° C./min. The sampleswere stirred at 5° C. for 12 hr, filtered or evaporated to dryness, andanalyzed by XRPD.

IPA Screening Procedure

4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilefree base (30 mg) was suspended in 1.2 ml of IPA at 50° C. and 1.1 eq ofcounterion solution was added. The samples were stirred at thistemperature for 30 min then cooled to 5° C. at 0.1° C./min. A stirringrate of 400 rpm was used throughout the experiments. After remaining at5° C. for up to 48 hr, the samples were rapidly heated to 50° C. heldfor 2 hr then cooled to room temperature. A control experiment with noadded counter-ion was also performed. The solids were filtered at roomtemperature, dried under vacuum (30° C.) for up to 24 hr and analyzed byXRPD.

1,4-Dioxane Screening Procedure

4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilefree base (30 mg) was dissolved in 1.5 ml of 1,4-dioxane at 50° C. togive a saturated solution and 1.1 eq of counterion solution was added.The samples were stirred at this temperature for 30 min then cooled to5° C. at 0.1° C./min. A stirring rate of 400 rpm was used throughout theexperiment. After remaining at 5° C. for 18 hr, the samples were rapidlyheated to 50° C. held for 2 hr then cooled to room temperature. Controlexperiment with no added counter-ion was performed. The solids werefiltered at room temperature, dried under vacuum (30° C.) for 18 hr andinitially analyzed by XRPD.

Dichloromethane Screening Procedure

4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilefree base (30 mg) was dissolved in 0.3 ml of dichloromethane at 35° C.and 1.1 eq of counterion solution was added. The samples were stirred atthis temperature for 30 min then cooled to 5° C. at 0.1° C./min. Astirring rate of 400 rpm was used throughout the experiment. Afterremaining at 5° C. for 18 hr, the solids were filtered at roomtemperature, dried under vacuum (30° C.) for 72 hr or air-dried andanalysed by XRPD. Control experiment with no added counterion wasperformed.

Summary of Results

XRPD results from the salt screen using procedures described above aresummarized in Tables 6-10. Every new pattern was labelled with thecounterion and a number, for example, methanesulfonic acid—MSA as MSA1,MSA 2.

TABLE 6 1,4-dioxane Counter-ion IPA screen screen DCM screen p-Toluenesulfonic pTSA 1 pTSA 1 pTSA 2 acid—pTSA Sulfuric acid—SO4 SO4 1 SO4 1SO4 1 (poor crystallinity) Methanesulfonic MSA 1 MSA 1 MSA 2 acid—MSA(poor crystallinity) Benzenesulfonic BSA 1 BSA 1 BSA 2 acid—BSAPhosphoric PHOA 1 PHOA 2 PHOA 3 acid—PHOA Malonic acid—MLNA MLNA 1 MLNA2 MLNA 1 L-Tartaric acid—TAR TAR 1 Amorphous Amorphous (poorcrystallinity) Fumaric acid—FUA FUA 1 FUA 2 FUA 3 (poor crystallinity)Citric acid—CA FB 1 CA 1 CA 2 (poor (poor crystallinity) crystallinity)L-Malic acid—MA MA 1 MA 2 MA 3 Benzoic acid—BA BA 1 BA 2 BA 2 Succinicacid—SUCA SUCA 1 SUCA 2 SUCA 3 Control FB 1 FB 2 FB 1

TABLE 7 Solubility in Stability post- FeSSiF (pH) XPRD storage at 40°XRPD of DSC pattern NMR and/or IC C./75% RH residue Endotherm 30.9° C.HCL 1 Consistent with Extra peaks 0.75 mg/ml (ΔH = −14 J/g) salt formobserved (4.89) Exotherm 168.2° C. 1 eq counterion Amorphous (ΔH = 11J/g) (titrimetric Endotherm 290.6° C. analysis) (ΔH = −84 J/g) Endotherm281.3° C. pTSA 1 1 eq counterion, No form change 0.87 mg/ml (ΔH = −74J/g) 0.008 eq dioxane, (4.85) pTSA 1 consistent with salt form Endotherm66.8° C. pTSA 2 1 eq counterion Slight change Not determined (ΔH = −48J/g) consistent with in pattern Endotherm 95.4° C. salt form (ΔH = −1J/g) Endotherm 139.7° C. (ΔH = −13 J/g) Endotherms 239.6° C. (ΔH = −86J/g) Endotherms 237.2° C. SO4 1 Consistent with No form change Notdetermined (ΔH = −79 J/g) salt form Endotherms 302.7° C. (ΔH = −236 J/g)Endotherms 231.1° C. Consistent with Not determined 0.72 mg/ml (ΔH = −59J/g) salt form (4.53) Endotherms 300.3° C. 1.1 eq counterion Amorphous(ΔH = −161 J/g) Endotherm 315.3° C. MSA 1 1 eq counterion, No formchange 1.01 mg/ml (ΔH = −103 J/g) 0.06 eq IPA, (4.86) Largely consistentwith amorphous salt form Endotherm 88.4° C. MSA 2 1 eq counterion Extrapeaks Not determined (ΔH = −2 J/g) consistent with observed Endotherm142.9° C. salt form (ΔH = −31 J/g) Endotherm 178.3° C. (ΔH = −70 J/g)Endotherms 254.2° C. (ΔH = −72 J/g) Endotherm 303.5° C. BSA 1 1 eqcounterion, No form change 0.84 mg/ml (ΔH = −109 J/g) 0.005 eq IPA,(4.86) consistent with BSA 1 - poor salt form crystallinity Endotherm89.4° C. BSA2 1 eq counterion Extra peaks Not determined (ΔH = −11 J/g)consistent with observed Endotherm 122.8° C. salt formation (ΔH = −24J/g) Endotherms 253° C. (ΔH = −132 J/g)

TABLE 8 Solubility in Stability post FeSSiF (pH) XPRD storage at 40°XRPD of DSC pattern NMR or IC C./75% RH residue Endotherm 235.6° C. PHOA1 0.04 eq IPA, No form change 0.74 mg/ml (ΔH = −132 J/g) 1.1 eqcounterion (4.83) - for (IC) TE-1184-31-01 consistent with Amorphoussalt formation Endotherm 187.7° C. PHOA 2 0.08 eq dioxane Pattern Notdetermined (ΔH = −16 J/g) consistent with change (poor Endotherm 194.1°C. salt formation crystallinity) (ΔH = −15 J/g) Endotherms 230.6° C. (ΔH= −102 J/g) Endotherms 61.9° C. PHOA 3 Peak shifts Reduced Notdetermined (ΔH = −57 J/g) consistent with crystallinity Endotherm 165.7°C. salt form (ΔH = −9 J/g) Endotherm 181.3° C. (ΔH −32 J/g) Endotherms227.3° C. (ΔH = −100 J/g) Endotherm 29.7° C. MLNA 1 1 eq counterion,Pattern Not determined (ΔH = −9 J/g) 0.02 eq IPA change (poor Endotherms172.2° C. consistent with crystallinity) (ΔH = −254 J/g) salt formationEndotherm 60.0° C. MLNA 2 Consistent with Pattern change Not determined(ΔH = −14 J/g) salt formation, Endotherm 110.9° C. 1 eq counterion, (ΔH= −19 J/g) 0.35 eq dioxane Exotherm 141.7° C. (ΔH = 15 J/g) Endotherm173.7° C. (ΔH = −196 J/g) Endotherm 28.9° C. MLNA 1 Not determinedPattern change Not determined (ΔH = −36 J/g) Endotherms 175.5° C. (ΔH =−213 J/g) Endotherm 31.0° C. TAR 1 Peak shifts Pattern Not determined(ΔH = −43 J/g) (poor consistent with change (poor Endotherm 122.7° C.crystallinity) salt form crystallinity) (ΔH = −11 J/g) Exotherm 158.8°C. (ΔH = 25 J/g) Endotherms 224.1° C. (ΔH = −159 J/g)

TABLE 9 Solubility in Stability post- FeSSiF (pH) XPRD storage at 40°XRPD of DSC pattern NMR or IC C./75% RH residue Endotherm 30.4° C. FUA 10.5 eq counterion, Pattern Not determined (ΔH = −44 J/g) 0.3 eq IPA,consistent change (poor Exotherm 154.5° C. with salt formation;crystallinity) (ΔH = 17 J/g) extra peaks at 3.57, Endotherm 238.1° C.3.74-3.78 ppm (ΔH = −149 J/g) Endotherm 36.9° C. FUA 2 0.5 eqcounterion, Increased Not determined (ΔH = −43 J/g) 0.2 eq dioxanecrystallinity Step changes (overlaps with 90° C. −140° C. broad signal)Endotherm 239.3° C. consistent with (ΔH = −160 J/g) salt formationEndotherm 29.5° C. FUA 3 0.4 eq counterion, Pattern change Notdetermined (ΔH = −33 J/g) consistent with Exotherm 180.8° C. saltformation, (ΔH = 25 J/g) extra peaks at Endotherms 243.5° C. 5.8,6.1-6.7 ppm (ΔH = −135 J/g) Endotherm 35.1° C. CA 1 Consistent withPattern change Not determined (ΔH = −7 J/g) salt formation, Endotherm126.3° C. counterion peaks (ΔH = −45.1 g) overlapping with Endotherm159.2° C. solvent and parent (ΔH = −109 J/g) signal Endotherm 35.5° C.CA 2 Consistent with No form change Not determined (ΔH = −4 J/g) saltformation, Endotherm 93.7° C. counterion peaks (ΔH = −21 J/g)overlapping with Endotherm 168.8° C. solvent and parent (ΔH = −156 J/g)signal Endotherm 29.8° C. MA 1 Peak shifts Amorphous 0.63 mg/ml (ΔH =−21 J/g) consistent with (4.89) Endotherm 157.3° C. salt formation,Amorphous (ΔH = −52 J/g) 0.5 eq IPA Exotherm 168.8° C. (ΔH = 44 J/g)Endotherm 212.8° C. (ΔH = −135 J/g) Multiple overlapping MA 2 Peakshifts Pattern Not determined events from 30° C. consistent with change(poor to 240° C. salt formation, crystallinity) 0.6 eq dioxane Multipleoverlapping MA 3 Peak shifts Pattern change Not determined events from30° C. consistent with to 240° C. salt formation

TABLE 10 Solubility in Stability post FeSSiF (pH) XPRD storage at 40°XRPD of DSC pattern NMR or IC C./75% RH residue Endotherm 231.9° C. BA 11 eq counterion, No significant 0.88 mg/ml (ΔH = −230 J/g) 0.32 eq IPA,change (4.85) - for consistent with TE-1184-31-02 salt formation Poorcrystallinity Exotherm 192.4° C. BA 2 1 eq counterion, No significantNot determined (ΔH = 5 J/g) 0.03 eq dioxane change Endotherm 234.9° C.consistent with (ΔH = −230 J/g) salt formation Endotherm 50.9° C. SECA 1Peak shifts Slight change 0.96 mg/ml (ΔH = −37 J/g) consistent with inpattern (4.89) Endotherm 177.8° C. salt formation Amorphous (ΔH = −71J/g) Endotherm 183.8° C. (ΔH = −31 J/g) Endotherm 41.0° C. SUCA 2 Peakshifts Pattern Not determined (ΔH = −101.4 J/g) consistent with change(poor Endotherm 97.5° C. salt formation crystallinity) (ΔH = −7 J/g)Endotherm 185.3° C. (ΔH = −32 J/g) Endotherm 201.4° C. (ΔH = −69 J/g)Endotherm 32.3° C. SUCA 3 Peak shifts Slight change Not determined (ΔH =−38 J/g) consistent with in pattern Endotherm 183.8° C. salt formation(ΔH = −61 J/g)

The screening experiments using three solvents (IPA, 1,4-dioxane andDCM) and twelve counterions gave twenty-six new solid forms.

One solid form (SO4-1) was isolated from screening using sulphuric acidin all three solvents. Experiments using tartaric acid only gave acrystalline form (TAR 1) with IPA and amorphous solids from 1, 4-dioxaneand DCM screens. At least two solid forms were obtained for each of theremaining ten counterions.

Representative samples for the twenty-six forms were further analysed byNMR/IC and DSC. The stability of the samples after storage at 40° C./75%RH was also assessed by XRPD. Solubility in FeSSIF media was determinedfor selected samples. Tables 6-10 show a summary of the preliminarycharacterization of the new forms. NMR data for all forms showed peakshifts consistent with salt formation and where applicable theequivalence of counterion and solvent was obtained. IC was employed todetermine the molar equivalence for inorganic counterions, sulphate andphosphate.

The onset temperature for DSC events gives indication of the thermalstability of the forms. pTSA 1, SO4 1, MSA 1, BSA 1, PHOA 1 and BA 1samples show good thermal stability. The onsets of DSC events aregreater than 200° C.; this data is consistent with non-solvatedcrystalline solids. The twenty remaining forms have thermal eventsoccurring from as low as ˜30° C. These may be solvated/hydrated forms.

The non-solvated forms remained unchanged by XRPD post storage at 40°C./75% RH while the other forms showed pattern changes. FeSSiFsolubility for these six forms (0.72-1.01 mg/ml) is comparable to theHCl salt value of 0.74 mg/ml.

The following have been identified as having improved stability andsolubility when compared to the HCl salt: pTSA 1, SO4-1, MSA 1, BSA1,PHOA 1, and BA 1.

Example 2: Preparation of4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrileBesylate

Step 1: Preparation of 2,5,6-trichloropyrimidin-4-ol

To a solution of 2,4,5,6-tetrachloropyrimidine (1 kg, 4.63 mol) in THF(6 L) was added 1N NaOH (6 L, 6.0 mol) dropwise, and the mixture wasstirred overnight at room temperature. The solution was acidified with1N HCl and extracted with DCM (3×). The combined organics were dried(Na₂SO₄) and concentrated in vacuo. The solids were slurried in Et₂O for30 min, filtered, washed with Et₂O and dried to give 635 g (69%) of thetitle compound. LCMS (C18 column, column size: 4.6*50 mm; mobile phase:20%-40%, Acetonitrile-Water-0.02% NH₄OAc): Rt=2.785 min; [M+H] Calc'dfor C₄HCl₃N₂O, 199. Found, 199.

Step 2: Preparation of 2,5,6-trichloro-3-methyl-3-hydropyrimidin-4-one

A solution of 2,5,6-trichloropyrimidin-4-ol (670 g, 3.38 mol) and K₂CO₃(560 g, 4.06 mol) in DMF (5 L) was stirred at room temperature for 15min and then cooled to 0° C. Iodomethane (528 g, 3.72 mol) was addeddropwise and the mixture was stirred at room temperature for 17 hr. Thereaction mixture was diluted with ethyl acetate, washed with brine,dried (Na₂SO₄) and concentrated in vacuo. The residue was purified bysilica gel chromatography (PE:EA, 10:1) to give 447 g (62%) of the titlecompound. ¹H NMR (400 MHz, CDCl₃): δ 3.74 (s, 3H). LCMS (C18 column;column size: 4.6*50 mm; mobile phase: 20%-95%, Acetonitrile-Water-0.02%NH₄OAc): Rt=2.986 min; [M+H] Calc'd for C₅H₃Cl₃N₂O, 213. Found, 213.

Step 3: Preparation ofN-[1-(5,6-dichloro-3-methyl-4-oxo(3-hydropyrimidin-2-yl))(4-piperidyl)](tert-butoxy)carboxamide

A solution of 2,5,6-trichloro-3-methyl-3-hydropyrimidin-4-one (532 g,2.51 mol), DIEA (648 g, 5.02 mol) and(tert-butoxy)-N-(4-piperidyl)carboxamide (502 g, 2.51 mol) in DMF (800mL) was heated to 120° C. for 1 hr. The solvent was removed in vacuo andthe residue was purified by silica gel chromatography (PE:EA, 1:1) togive 751 g (80%) of the title compound. ¹H NMR (400 MHz, CDCl₃): δ 1.45(s, 9H), 1.50-1.58 (m, 2H), 2.06-2.10 (m, 2H), 2.98-3.05 (m, 2H), 3.48(s, 3H), 3.53-3.56 (m, 2H), 3.70 (s, 1H), 4.52 (s, 1H). LCMS (C18column; column size: 4.6*50 mm; mobile phase: 20%-95%,Acetonitrile-Water-0.02% NH₄OAc): Rt=4.006 min; [M+H] Calc'd forC₁₅H₂₂Cl₂N₄O₃, 377. Found, 321 (MW-tBu).

Step 4: Preparation of tert-butyl1-(5-chloro-4-(3-fluoro-4-cyanophenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)piperidin-4-ylcarbamate

To a solution ofN-[1-(5,6-dichloro-3-methyl-4-oxo(3-hydropyrimidin-2-yl))(4-piperidyl)](tert-butoxy)carboxamide (150 g, 0.40 mol) in ACN (4 L),under N₂ atmosphere, was added 3-fluoro-4-cyanophenylboronic acid (65.8g, 0.40 mol), Pd(Ph₃P)₄ (9.3 g, 8 mmol) and 0.4 N Na₂CO₃ (2 L, 0.80mol). The mixture was stirred at 85° C. for 2 hr and was allowed to coolto room temperature. Water (2 L) was added, and the aqueous mixture wasextracted with ethyl acetate (3×). The organics were combined, washedwith water, washed with brine, dried (Na₂SO₄) and concentrated. Theresidue was purified by silica gel chromatography (PE:EA, 3:1) to give95 g (57%) of the title compound. ¹H NMR (400 MHz, CDCl₃): δ 1.45 (s,9H), 1.54-1.61 (m, 2H), 2.05-2.13 (m, 2H), 2.99-3.08 (m, 2H), 3.53-3.58(s, 5H), 3.70 (s, 1H), 4.54 (d, J=6.0 Hz, 1H), 7.68-7.80 (m, 3H). LCMS(C18 column; column size: 4.6*50 mm; mobile phase: 5%-95%,Acetonitrile-Water-0.1% TFA): Rt=4.443 min; [M+H] Calc'd forC₂₂H₂₅ClFN₅O₃, 462. Found, 462.

Step 5: Preparation of tert-butylN-[1-[4-(4-cyano-3-fluorophenyl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxopyrimidin-2-yl]piperidin-4-yl]carbamate

A mixture of(tert-butoxy)-N-{1-[5-chloro-6-(4-cyano-3-fluorophenyl)-3-methyl-4-oxo-(3-hydropyrimidin-2-yl)]-(4-piperidyl)}carboxamide(295.8 g, 640 mmol), 3-fluoro-4-methoxy benzeneboronic acid (217.7 g,1281 mmol), Pd(Ph₃P)₄ (18.5 g, 16.0 mmol) and K₂CO₃ (265.5 g, 1921 mmol)in degassed dioxane:H₂O (3:1, 3550 mL:887 mL) was stirred, under N₂atmosphere, at 80° C. until reaction completion (at least 2 hr). Thereaction mixture was allowed to cool to room temperature. Water (11 L)was added, and the slurry was stirred for 1 hr. The slurry was filtered,and the solids were washed with water (4 L) and washed with MeOH:Water(1:1, 4 L). The cake was stirred in MeOH (4 L) for 10 min. The slurrywas filtered, and the solids were rinsed with MeOH (4 L) and washed withMTBE (4 L). The solids were taken in DCM (16 L), and the reactionmixture was stirred for 15 min. 2-mercaptoethyl ethyl sulfide silica(400 g) was then added, and the reaction mixture was stirred at roomtemperature, under N₂ atmosphere, for at least 1 hr. The reactionmixture was filtered through Celite in a frit filter, and the solidswere rinsed with DCM (1 L). The volume of DCM filtrate was reduced tonear dryness. MeOH (4 L) was added, and the solvent was reduced todryness. The solids were taken in MeOH (4 L), and the slurry was cooledto 15° C. The slurry was filtered, and the cake was washed with MeOH(0.7 L), washed with MTBE (0.7 L) and dried to constant weight undervacuum at 45° C. to give 324.6 g (91.9%) of the title compound. ¹H NMR(400 MHz, CDCl₃): δ 1.46 (s, 9H), 1.60 (d, J=10.11 Hz, 2H), 2.11 (d,J=11.62 Hz, 2H), 3.06 (t, J=12.00 Hz, 2H), 3.54 (s, 3H), 3.60 (d,J=13.64 Hz, 2H), 3.72 (br. s., 1H), 3.88 (s, 3H), 4.52 (br. s., 1H),6.79-6.89 (m, 2H), 6.97 (d, J=12.38 Hz, 1H), 7.13 (d, J=8.34 Hz, 1H),7.31 (d, J=9.85 Hz, 1H), 7.42 (br. s., 1H). LCMS (C18 column; columnsize: 4.6*50 mm; mobile phase: 5%-95%, Acetonitrile-Water-0.1% TFA):RT=6.979; [M+H] Calc'd for C₂₉H₃₁F₂N₅O₄, 552. Found, 552.

Step 6: Preparation of4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluoro-benzonitrile,Besylate Salt

A solution of tert-butylN-[1-[4-(4-cyano-3-fluorophenyl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxopyrimidin-2-yl]piperidin-4-yl]carbamate(5.0 g, 9.06 mmol) and benzenesulfonic acid monohydrate (1.9 g, 10.9mmol) in formic acid (41.5 mL) was stirred at room temperature untilreaction completion. The solution was filtered through a 0.45 μm filter.Water (25 mL) was added to the formic acid solution. Seeds of4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluoro-benzonitrile,besylate salt (0.05 g) were introduced, and the solution was aged for 30min. Water (up to 50 mL) was added to the mixture over 6 hr. The batchwas then allowed to age for at least 12 hr. The batch was filtered, andthe cake was washed with 80/20 Water/Formic Acid (v/v) and dried at40-50° C. in a vacuum oven with a nitrogen bleed to give the (5.25 g,95%) of title compound. ¹H NMR (400 MHz, CD₃OD): δ 1.69 (q, 2H, J=11.4Hz), 2.00 (d, 2H, J=10.2 Hz), 2.99 (t, 2H, J=12.3 Hz), 3.31 (bs, 1H),3.42 (s, 3H), 3.72 (d, 2H, J=13.2 Hz), 3.81 (s, 3H), 6.78 (d, 1H, J=8.4Hz), 7.01-7.06 (m, 1H), 7.04-7.06 (m, 1H), 7.19 (dd, 1H, 1.2 Hz), 7.32(m, 2H), 7.32 (m, 1H), 7.46 (dd, 1H, J=10.5, 1.2 Hz), 7.61 (m, 2H), 7.82(dd, 1H, J=8.1, 7.2 Hz), 7.92 (bs, 1H), 7.92 (bs, 2H). LCMS (Column:Agilent Zorbax SB-C8, 4.6×50 mm, 3.5 um particle size; mobile phase:5%-95%, Acetonitrile-Water-0.1% TFA): RT=3.854; [M+H] Calc'd forC₂₄H₂₃F₂N₅O₂, 452. Found, 452. A second recrystallization from 80/20water/formic acid was performed as described above to provide materialgreater than 99% pure as determined by LC analysis.

The DSC thermogram of the title compound is provided in FIG. 2.

The XRPD pattern of Form 1 of the title compound is provided in FIG. 1.Characteristic diffraction peaks include 4.9° 2-Theta, 9.7° 2-Theta,13.4° 2-Theta, 18.0° 2-Theta and 18.5° 2-Theta.

Example 3: Preparation of Amorphous4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrileBesylate

Amorphous4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrilebesylate was prepared by ball milling Form 1 crystalline4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrilebesylate for 99 min at 30 Hz. The resulting material was observed to beamorphous by XRPD analysis as shown in FIG. 3.

Example 4: Pharmaceutical Composition—Capsule Formulation

In one embodiment, capsule formulations of crystalline4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt for administration to humans are prepared with thefollowing ingredients:

TABLE 11 Components of Capsule Formulation Quantity Quantity per Sizeper Size Component Function 4 Capsule 1 Capsule crystalline4-[2-(4-amino- Active 0.5 to 5 to piperidin-1-yl)-5-(3-fluoro- 100 mg500 mg 4-methoxy-phenyl)-1-methyl-6- oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrile besylate salt Hypromellose, USP Capsule 1capsule 1 capsule Shell

The process to prepare crystalline4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt in a capsule is as follows: Weigh the required amount of4-[2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl]-2-fluorobenzonitrilebesylate salt, add into the appropriate size capsule, and close capsule.

The examples and embodiments described herein are illustrative andvarious modifications or changes suggested to persons skilled in the artare to be included within this disclosure. As will be appreciated bythose skilled in the art, the specific components listed in the aboveexamples may be replaced with other functionally equivalent components,e.g., diluents, binders, lubricants, fillers, and the like.

What is claimed is:
 1. A hydrochloride salt of4-[2-(4-amino-piperidin-1-yl)-5-(3-fluoro-4-methoxy-phenyl)-1-methyl-6-oxo-1,6-dihydro-pyrimidin-4-yl]-2-fluorobenzonitrile.2. The hydrochloride salt of claim 1, wherein the hydrochloride salt isamorphous.
 3. The hydrochloride salt of claim 1, wherein thehydrochloride salt is crystalline and has at least one of the followingproperties: (a) a DSC (differential scanning calorimetry) thermogramwith an endotherm at about 30.9° C.; (b) a DSC (differential scanningcalorimetry) thermogram with an endotherm at about 290.6° C.; or (c) aDSC (differential scanning calorimetry) thermogram with an exotherm atabout 168.2° C.
 4. The hydrochloride salt of claim 3, wherein thehydrochloride salt is crystalline and has a DSC thermogram with anendotherm at about 30.9° C. and about 290.6° C.
 5. The hydrochloridesalt of claim 3, wherein the hydrochloride salt is crystalline and has aDSC thermogram with an endotherm at about 30.9° C. and about 290.6° andan exotherm at about 168.2° C.
 6. The hydrochloride salt of claim 3,wherein the hydrochloride salt is hygroscopic with about 14% uptake at90% RH by GVS (gravimetric vapor sorption).
 7. The hydrochloride salt ofclaim 3, wherein the hydrochloride salt has FeSSIF (fed state simulatedintestinal fluid) solubility at about 0.75 mg/mL.
 8. The hydrochloridesalt of claim 7, wherein the hydrochloride salt has a pH of about 4.89.9. A pharmaceutical composition comprising a hydrochloride salt of claim1 and at least one pharmaceutically acceptable carrier, diluent, orexcipient.
 10. The pharmaceutical composition of claim 9, where thepharmaceutical composition is in an oral dosage form.
 11. Thepharmaceutical composition of claim 10, where the oral dosage form is ina table, capsule, or pill.